Compound containing a labile disulfide bond

ABSTRACT

A labile disulfide-containing compound under physiological conditions containing a labile disulfide bond and a transduction signal.

[0001] This application is a continuation-in-part of Ser. No. 09/312,351filed on May 14, 1999.

BACKGROUND

[0002] Bifunctional molecules, commonly referred to as crosslinkers, areused to connect two molecules together. Bifunctional molecules cancontain homo or heterobifunctionality. The disulfide linkage (RSSR′) maybe used within bifunctional molecules. The reversibility of disulfidebond formation makes them useful tools for the transient attachment oftwo molecules. Disulfides have been used to attach a bioactive compoundand another compound (Thorpe, P. E. J. Natl. Cancer Inst. 1987, 79,1101). The disulfide bond is reduced thereby releasing the bioactivecompound. Disulfide bonds may also be used in the formation of polymers(Kishore, K., Ganesh, K. in Advances in Polymer Science, Vol. 21,Saegusa, T. Ed., 1993).

[0003] There are many commercially available reagents for the linkage oftwo molecules by a disulfide bond. Additionally there are bifunctionalreagents that have a disulfide bond present. Typically, these reagentsare based on 3-mercaptopropionic acid, i.e. dithiobispropionate.However, the rate at which these bonds are broken under physiologicalconditions is slow. For example, the half life of a disulfide derivedfrom dithiobispropionimidate, an analog of 3-mercaptopropionic acid, is27 hours in vivo (Arpicco, S., Dosio, F., Brusa, P., Crosasso, P.,Cattel, L. Bioconjugate Chem. 1997, 8, 327.). A stable disulfide bond isoften desirable, for example when purification of linked molecules orlong circulation in vivo is needed. For this reason, attempts have beenmade to make the disulfide less susceptible to cleavage.

[0004] It has been demonstrated that both stability, measured asreduction potential, and rate, measured as rate constants, of disulfidereduction are both related to the acidity of the thiols which constitutethe disulfide. Additional factors that may affect the rate of reductionare steric interactions, and intramolecular disulfide cleavage. Lookingat the difference in the rates for the reactions RSH+R′SSR′→RSSR′+R′SHand RSH+R″SSR″→RSSR″+R″SH, it has been demonstrated that logk″/k′=β(pK_(a) ^(R′)-pK_(a) ^(R″)), where k′ and k″ are the rateconstant for the reactions with R′SSR′ and R″SSR″ respectively, pK_(a)^(R′) and pK_(a) ^(R″) are the acidities of the thiol groups R′SH andR″SH, and β is a constant determined empirically to be 0.72. From thisequation, one would predict that the reduction of a disulfide composedfrom relatively acidic thiols would be reduced more quickly than onecomposed of less acidic thiols. In support of this observation, it hasbeen demonstrated that the disulfides cystine (pK_(a) 8.3) and cystamine(pK_(a) 8.2) are reduced 3-15 times faster than oxidized glutathione(pK_(a) 8.9) (Bulaj, G., Kortemme, T., Goldenberg, D. P. Biochemistry1998, 37, 8965).

[0005] It has been demonstrated that both stability (thermodynamics),measured as reduction potential (Keire D. A. J. Org. Chem. 1992, 57,123), and rate (kinetics), measured as rate constants, of disulfidereduction are both related to the acidity of the thiols which constitutethe disulfide (Szajewski, R. P., Whitesides, G. M. J. Am. Chem. Soc.1980, 102, 2011). The increase in acidity of a thiol is dependent uponone or more of the following structural factors: the presence ofelectron withdrawing groups which stabilize the thiolate through sigmaand pi bonds (inductive effect), the presence of electron withdrawinggroups that stabilize the thiolate through space or solvent (fieldeffects), pi bonds which allow the negative charge to be placed on otheratoms (resonance stabilization), and hydrogen bond donating groupswithin the molecule that can interact internally with the thiolate. Forexample, cysteine has an amino group two atoms from the thiol, which ismore electron withdrawing than the amide nitrogen that is two atoms fromthe thiol in glutathione. As a consequence of this difference inelectron withdrawing groups, the thiol of cysteine is 0.6 pK units moreacidic than glutathione, and as mentioned previously, cystine is reduced3-15 times faster than oxidized glutathione. Another example of arelatively acidic thiol is 5-thio-2-nitrobenzoic acid, pK_(a) 5. Itsacidity is due to resonance stabilization and inductive effects. Itsdisulfide is rapidly reduced by all standard alkyl thiols and itscolored thiolate makes it a convenient assay for thiol concentration.

SUMMARY

[0006] Described in a preferred embodiment is a process for the deliveryof a compound to a cell, comprising associating a compound, containing adisulfide bond that can be cleaved under physiological conditions, witha polymer, then delivering the polymer to the cell. The polymer maycomprise a first polymer and a second polymer. The first polymer and thesecond polymer may comprise nucleic acids, proteins, genes, antisensepolymers, DNA/RNA hybrids, or synthetic polymers.

[0007] In another preferred embodiment, a biologically active compoundis associated with a disulfide-containing compound, comprising: thedisulfide-containing compound having a labile disulfide bond that isselected from the group consisting of (a) a disulfide bond that iscleaved more rapidly than oxidized glutathione and (b) a disulfide bondconstructed from thiols in which one of the constituent thiols has alower pKa than glutathione and (c) a di sulfide bond that is activatedby intramolecular attack from a free thiol.

[0008] In another preferred embodiment, a compound is provided forinserting into an organism, comprising: the compound having a disulfidebond that is labile under physiologic conditions selected from the groupconsisting of (a) a disulfide bond that is cleaved more rapidly thanoxidized glutathione and (b) a disulfide bond constructed from thiols inwhich one of the constituent thiols has a lower pKa than glutathione and(c) a disulfide bond that is activated by intramolecular attack from afree thiol.

[0009] In another preferred embodiment, a process is provided forforming a compound having a labile disulfide bond for use with anorganism, comprising: forming the compound having a disulfide bondselected from the group consisting of (i) a disulfide bond that iscleaved more rapidly than oxidized glutathione, and (ii) a disulfidebond constructed from thiols in which one of the constituent thiols hasa lower pKa than glutathione, and (iii) a disulfide bond that isactivated by intramolecular attack from a free thiol; inserting thecompound into the organism.

[0010] In another preferred embodiment, a process is described forcompacting a nucleic acid for delivery to a cell, comprising associatinga polymer containing a disulfide bond with a nucleic acid and deliveringthe nucleic acid to the cell.

[0011] In another preferred embodiment, a process is described forcompacting a nucleic acid for delivery to a cell comprising associatinga polymer with the nucleic acid, then associating a compound containinga disulfide bond that can be cleaved under physiological conditions withthe nucleic acid polymer complex, then delivering the complex to a cell.

[0012] In another preferred embodiment, a process is described forcompacting a nucleic acid for delivery to a cell, comprising associatinga polymer containing a disulfide bond with a nucleic acid, thenassociating another polymer with the disulfide containingpolymer—nucleic acid complex, then delivering the complex to the cell.

[0013] In another preferred embodiment, a process is described forcompacting a nucleic acid for delivery to a cell comprising associatinga polymer with the nucleic acid, then associating a compound containinga disulfide bond that can be cleaved under physiological conditions withthe nucleic acid polymer complex, then associating another polymer withthe complex, then delivering the complex to a cell.

[0014] In another preferred embodiment, a compound is described whichcontains a disulfide bond that can be cleaved under physiologicalconditions and possesses heterobifunctional or homobifunctional groups.Such a compound can be described as a disulfide containing bifunctionalmolecule.

A₁—S—S—A₂

[0015] More particularly, a compound that contains an aliphaticdisulfide bond with one or more electronegative (electron withdrawinggroups) substituted alpha or beta to one or both of the sulfur atoms.These groups serve to lower the pK_(a) of the constituent thiols.

[0016] Where R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈—at least one of which is anelectronegative atom or functionality such as OH, OR (an ether),NH₂,(also secondary, tertiary, and quaternary amines), SO₃ ⁻, COOH, COOR(an ester), CONH₂, CONR₂ (substituted amide), a halogen (F, Cl, Br, O),NO₂. L is defined as a linker or spacer group that provides a connectionbetween the disulfide and the reactive heterobifunctional orhomobifunctional groups, A₁ and A₂. L may or may not be present and maybe chosen from a group that includes alkanes, alkenes, alkynes, esters,ethers, glycerol, amide, urea, saccharides, polysaccharides, heteroatomssuch as oxygen, sulfur, or nitrogen. The spacer may be charge positive,charge negative, charge neutral, or zwitterionic. A₁ and A₂ are reactivegroups they may be identical as in a homobifunctional bifunctionalmolecule, or different as in a heterobifunctional bifunctional molecule.In a preferred embodiment, the disulfide compounds contain reactivegroups that can undergo acylation or alkylation reactions. Such reactivegroups include (but not limited to) isothiocyanate, isocyanate, acylazide, acid halide, O-acyl urea, N-hydroxysuccinimide esters,succinimide esters, amide, urea, sulfonyl chloride, aldehyde, ketone,ether, epoxide, carbonate, alkyl halide, imidoester, carboxylate,alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) oranhydrides.

[0017] If functional group A1, A2 is an amine then A1, A2 can react with(but not restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionA1, A2 is an amine, then an acylating or alkylating agent can react withthe amine.

[0018] If functional group A1, A2 is a sulflhydryl then A1, A2 can reactwith (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0019] If functional group A1, A2 is carboxylate then A1, A2 can reactwith (but not restricted to) a diazoacetate, alcohol, thiol or an amineonce the acid has been activated.

[0020] If functional group A1, A2 is an hydroxyl then A1, A2 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0021] If functional group A1, A2 is an aldehyde or ketone then A1, A2can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0022] If functional group A1, A2 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then A1, A2can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0023] If functional group A1, A2 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) thenA1, A2 can react with (but not restricted to) a sulfhydryl.

[0024] If functional group A1, A2 is an aldehyde, ketone, epoxide,oxirane, or an amine in which carbonyldiimidazole or N,N′-disuccinimidylcarbonate is used, then A1, A2 can react with (but not restricted to) ahydroxyl.

[0025] If functional group A1, A2 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then A1, A2 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

[0026] Additionally, a compound which contains an aromatic disulfidebond in which the sulfur atom is bonded directly to the aromatic ring.The ring may contain 5 or more atoms.

[0027] R₁-R₄, R₆-R₉—The substitution pattern on the ring may be variedto alter the reduction potential of the disulfide bond. The substiuentsmay be selected from the group that includes but is not limited to OH,OR (an ether), NH₂,(also secondary, tertiary, and quaternary amines),SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), ahalogen (F, Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain,saturated, or unsaturated aliphatic group). L is defined as a linker orspacer group that provides a connection between the disulfide and thereactive heterobifinctional or homobifunctional groups. L may or may notbe present and may be chosen from a group that includes alkanes,alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides,heteroatoms such as oxygen, sulfur, or nitrogen. The spacer may becharge positive, charge negative, charge neutral, or zwitterionic. R₅,R₁₀—are reactive groups they may be identical as in a homobifunctionalbifunctional molecule, or different as in a heterobifunctionalbifunctional molecule. In a preferred embodiment, the disulfidecompounds contain reactive groups that can undergo acylation oralkylation reactions. Such reactive groups include isothiocynanate,isocynanate, acyl azide, N-hydroxysuccinimide esters, succinimideesters, sulfonyl chloride, aldehyde, epoxide, carbonate, imidoester,carboxylate, alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene)or succinic anhydride.

[0028] If functional group R₅, R₁₀ is an amine then R₅, R₁₀ can reactwith (but not restricted to) an activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,epoxide, carbonate, imidoester, amide, carboxylate, or alkylphosphate,arylhalides (difluoro-dinitrobenzene) or anhydrides. In other terms whenfunction R5, R10 is an amine, then an acylating or alkylating agent canreact with the amine.

[0029] If functional group R5, R10 is a sulfhydryl then R5, R10 canreact with (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0030] If functional group R5, R10 is carboxylate then R5, R10 can reactwith (but not restricted to) a diazoacetate, alcohol, thiol or an amineonce the acid has been activated.

[0031] If functional group R5, R10 is an hydroxyl then R5, R10 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0032] If functional group R5, R10 is an aldehyde or ketone then R5, R10can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0033] If functional group R5, R10 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R5, R10can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0034] If functional group R5, R10 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) thenR5, R10 can react with (but not restricted to) a sulfhydryl. Iffunctional group R5, R10 is an aldehyde, ketone, epoxide, oxirane, or anamine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonate isused, then R5, R10 can react with (but not restricted to) a hydroxyl.

[0035] If functional group R5, R10 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then R5, R10 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

[0036] Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring. The heterocyclic ring may bearomatic or aliphatic. The heterocyclic ring may contain 5 or more atomsof which 1 or more is a heteroatom (O, N, S, P), and the rest beingcarbon atoms

[0037] H is a heteroatom selected from the group including sulfur,oxygen, nitrogen, or phosphorus. R₁-R₃, R₅-R₇ are substiuents that maybe selected from the group that includes but is not limited to OH, OR(an ether), NH₂,(also secondary, tertiary, and quaternary amines), SO₃⁻, COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), a halogen(F, Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain,saturated, or unsaturated aliphatic group). The substitution pattern onthe aromatic ring may be varied to alter the reduction potential of thedisulfide bond. L is defined as a linker or spacer group that provides aconnection between the disulfide and the reactive heterobifunctional orhomobifunctional groups. L may or may not be present and may be chosenfrom a group that includes alkanes, alkenes, esters, ethers, glycerol,amide, saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,or nitrogen. The spacer may be charge positive, charge negative, chargeneutral, or zwitterionic. R₄, R₈ are reactive groups they may beidentical as in a homobifunctional bifunctional molecule, or differentas in a heterobifunctional bifunctional molecule. In a preferredembodiment, the disulfide compounds contain reactive groups that canundergo acylation or alkylation reactions. Such reactive groups includeisothiocynanate, isocynanate, acyl azide, N-hydroxysuccinimide esters,succinimide esters, sulfonyl chloride, aldehyde, epoxide, carbonate,imidoester, carboxylate, alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) or succinic anhydride.

[0038] If functional group R4, R8 is an amine then R4, R8 can react with(but not restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro- dinitrobenzene) or anhydrides. In other terms when functionR4, R8 is an amine, then an acylating or alkylating agent can react withthe amine.

[0039] If functional group R4, R8 is a sulfhydryl then R4, R8 can reactwith (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0040] If functional group R4, R8 is carboxylate then R4, R8 can reactwith (but not restricted to) a diazoacetate, alcohol, thiol or an amineonce the acid has been activated.

[0041] If functional group R4, R8 is an hydroxyl then R4, R8 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0042] If functional group R4, R8 is an aldehyde or ketone then R4, R8can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0043] If functional group R4, R8 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R4, R8can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0044] If functional group R4, R8 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) thenR4, R8 can react with (but not restricted to) a sulfhydryl.

[0045] If functional group R4, R8 is an aldehyde, ketone, epoxide,oxirane, or an amine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonate is used, then R4, R8 can react with (but notrestricted to) a hydroxyl.

[0046] If functional group R4, R8 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then R4, R8 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

[0047] Additionally, a compound which contains a disulfide bond that isconnected directly to a ring system(aromatic or non-aromatic) throughone of the sulfur atoms and to a aliphatic carbon through the othersulfur atom. The cyclic ring may contain 5 or more atoms.

[0048] R₁-R₄ are substiuents selected from the group that includes butis not limited to H, OH, OR (an ether), NH₂,(also secondary, tertiary,and quaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂(substituted amide), a halogen (F, Cl, Br, I), NO₂, CH₃ (or longerbranched or straight chain, saturated, or unsaturated aliphatic group).The substitution pattern on the aromatic ring may be varied to alter thereduction potential of the disulfide bond. R₆-R₉ are substiuentsselected from the group that includes but is not limited to H, OH, OR(an ether), NH₂,(also secondary, tertiary, and quaternary amines), SO₃^(b−), COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), ahalogen (F, Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain,saturated, or unsaturated aliphatic group). L is defined as a linker orspacer group that provides a connection between the disulfide and thereactive heterobifunctional or homobifunctional groups. L may or may notbe present and may be chosen from a group that includes alkanes,alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides,heteroatoms such as oxygen, sulfur, or nitrogen. The spacer may becharge positive, charge negative, charge neutral, or zwitterionic. R₅,and R₁₀ are reactive groups that may be identical as in ahomobifunctional bifunctional molecule, or different as in aheterobifunctional bifunctional molecule. In a preferred embodiment, thedisulfide compounds contain reactive groups that can undergo acylationor alkylation reactions. Such reactive groups include isothiocynanate,isocynanate, acyl azide, N-hydroxysuccinimide esters, succinimideesters, sulfonyl chloride, aldehyde, epoxide, carbonate, imidoester,carboxylate, alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene)or succinic anhydride.

[0049] If functional group R5, R10 is an amine then R5, R10 can reactwith (but not restricted to) an activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,epoxide, carbonate, imidoester, amide, carboxylate, or alkylphosphate,arylhalides (difluoro-dinitrobenzene) or anhydrides. In other terms whenfunction R5, R10 is an amine, then an acylating or alkylating agent canreact with the amine.

[0050] If functional group R5, R10 is a sulfhydryl then R5, R10 canreact with (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0051] If functional group R5, R10 is carboxylate then R5, R10 can reactwith (but not restricted to) a diazoacetate, alcohol, thiol or an amineonce the acid has been activated.

[0052] If functional group R5, R10 is an hydroxyl then R5, R10 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0053] If functional group R5, R10 is an aldehyde or ketone then R5, R10can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0054] If functional group R5, R10 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R5, R10can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0055] If functional group R5, R10 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) thenR5, R10 can react with (but not restricted to) a sulfhydryl.

[0056] If functional group R5, R10 is an aldehyde, ketone, epoxide,oxirane, or an amine in which carbonyldiimidazole or N,N′-disuccinimidylcarbonate is used, then R5, R10 can react with (but not restricted to) ahydroxyl.

[0057] If functional group R5, R10 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then R5, R10 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

[0058] Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring system through one of thesulfur atoms and to a aliphatic carbon through the other sulfur atom.The heterocyclic ring may contain 5 or more atoms of which 1 or more isa heteroatom (O, N, S, P) or combinations of heteroatoms, and the rest

[0059] being carbon atoms.

[0060] H is a heteroatom selected from the group including sulfur,oxygen, nitrogen, or phosphorus. Where R₁, R₂, R₃, R₄, R₅, R₆, R₇,R₈,R₉, R₁₀, R₁₁, R₁₂, R₁₄—at least one of which is an electronegativeatom or functionality such as OH, OR (an ether), NH₂, (also secondary,tertiary, and quaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂,CONR₂ (substituted amide), a halogen (F, Cl, Br, I), NO₂. L is definedas a linker or spacer group that provides a connection between thedisulfide and the reactive heterobifunctional or homobifunctionalgroups, A₁ and R₉. L may or may not be present and may be chosen from agroup that includes alkanes, alkenes, alkynes, esters, ethers, glycerol,amide, urea, saccharides, polysaccharides, heteroatoms such as oxygen,sulfur, or nitrogen. The spacer may be charge positive, charge negative,charge neutral, or zwitterionic. A₁ and R9 are reactive groups they maybe identical as in a homobifunctional bifunctional molecule, ordifferent as in a heterobifunctional bifunctional molecule. In apreferred embodiment, the disulfide compounds contain reactive groupsthat can undergo acylation or alkylation reactions. Such reactive groupsinclude (but not limited to) isothiocynanate, isocynanate, acyl azide,acid halide, 0-acyl urea, N-hydroxysuccinimide esters, succinimideesters, amide, urea, sulfonyl chloride, aldehyde, ketone, ether,epoxide, carbonate, alkyl halide, imidoester, carboxylate,alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) oranhydrides.

[0061] If functional group A1, R9 is an amine then A1, R9 can react with(but not restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionA1, R9 is an amine, then an acylating or alkylating agent can react withthe amine.

[0062] If functional group A1, R9 is a sulfhydryl then A1, R9 can reactwith (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0063] If functional group A1, R9 is carboxylate then A1, R9 can reactwith (but not restricted to) a diazoacetate, alcohol, thiol or an amineonce the acid has been activated.

[0064] If functional group A1, R9 is an hydroxyl then A1, R9 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0065] If functional group A1, R9 is an aldehyde or ketone then A1, R9can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0066] If functional group A1, R9 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then A1, R9can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0067] If functional group A1, R9 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) thenA1, R9 can react with (but not restricted to) a sulfhydryl.

[0068] If functional group A1, R9 is an aldehyde, ketone, epoxide,oxirane, or an amine in which carbonyldiimidazole or N,N′-disuccinimidylcarbonate is used, then A1, R9 can react with (but not restricted to) ahydroxyl.

[0069] If functional group A1, R9 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then A1, R9 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

[0070] Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring system (aromatic ornon-aromatic) through one of the sulfur atoms and to an aromatic ringsystem through the other sulfur atom. The heterocyclic ring may contain5 or more atoms of which 1 or more is a heteroatom (O, N, S, P) orcombinations of heteroatoms, and the rest being carbon atoms.

[0071] H is a heteroatom selected from the group including sulfur,oxygen, nitrogen, or phosphorus. Where R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,R₁₀, R₁₁, R₁₂, R₁₃—at least one of which is an electronegative atom orfunctionality such as OH, OR (an ether), NH₂, (also secondary, tertiary,and quaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂(substituted amide), a halogen (F, Cl, Br, I), NO₂. L is defined as alinker or spacer group that provides a connection between the disulfideand the reactive heterobifunctional or homobifunctional groups, R₉ andR₁₄. L may or may not be present and may be chosen from a group thatincludes alkanes, alkenes, alkynes, esters, ethers, glycerol, amide,urea, saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,or nitrogen. The spacer may be charge positive, charge negative, chargeneutral, or zwitterionic. R₉ and R₁₄ are reactive groups they may beidentical as in a homobifunctional bifunctional molecule, or differentas in a heterobifunctional bifunctional molecule. In a preferredembodiment, the disulfide compounds contain reactive groups that canundergo acylation or alkylation reactions. Such reactive groups include(but not limited to) isothiocynanate, isocynanate, acyl azide, acidhalide, O-acyl urea, N-hydroxysuccinimide esters, succinimide esters,amide, urea, sulfonyl chloride, aldehyde, ketone, ether, epoxide,carbonate, alkyl halide, imidoester, carboxylate, alkylphosphate,arylhalides (e.g. difluoro-dinitrobenzene) or anhydrides.

[0072] If functional group R9, R14 is an amine then R9, R14 can reactwith (but not restricted to) an activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, alkyl halide, acid halide,N-hydroxysuccinimide ester, sulfonyl chloride, aldehyde, ketone,epoxide, carbonate, imidoester, amide, carboxylate, or alkylphosphate,arylhalides (difluoro-dinitrobenzene) or anhydrides. In other terms whenfunction R9, R14 is an amine, then an acylating or alkylating agent canreact with the amine.

[0073] If functional group R9, R14 is a sulfhydryl then R9, R14 canreact with (but not restricted to) a haloacetyl derivative, activatedcarboxylic acid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

[0074] If functional group R9, R14 is carboxylate then R9, R1 4 canreact with (but not restricted to) a diazoacetate, alcohol, thiol or anamine once the acid has been activated.

[0075] If functional group R9, R14 is an hydroxyl then R9, R14 can reactwith (but not restricted to) an activated carboxylic acid, epoxide,oxirane, or an amine in which carbonyldiimidazole is used.

[0076] If functional group R9, R14 is an aldehyde or ketone then R9, R14can react with (but not restricted to) an hydrazine, hydrazidederivative, amine (to form a Schiff Base that may or may not besubsequently reduced by reducing agents such as NaCNBH₃), or a diol toform an acetal or ketal.

[0077] If functional group R9, R14 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R9, R14can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

[0078] If functional group R9, R14 an activated carboxylic acid,haloacetyl derivative, maleimide, aziridine derivative, acryloylderivative, fluorobenzene derivatives, or disulfide derivative (such asa pyridyl disulfide or 5-thio-2-nitrobenzoic acid {TNB} derivatives)then R9, R14 can react with (but not restricted to) a sulfhydryl.

[0079] If functional group R9, R14 is an aldehyde, ketone, epoxide,oxirane, or an amine in which carbonyldiimidazole or N,N′-disuccinimidylcarbonate is used, then R9, R14 can react with (but not restricted to) ahydroxyl.

[0080] If functional group R9, R14 is a hydrazine, hydrazide derivative,or amine (primary or secondary) then R9, R14 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

DETAILED DESCRIPTION

[0081] Counterintuitive to previous efforts to synthesize bifunctionalmolecules with stabile disulfides, the object of the current inventionis to synthesize labile disulfide molecules. In vivo, disulfides areprimarily reduced by the cysteine-based thiol glutathione(γ-glutamylcystylglycine), which is present in millimolar concentrationsin the cell. To increase the lability of the disulfide bond in abifunctional molecule and its construct, we have synthesized severaldisulfide bond-containing bifunctional molecules that are more rapidlyreduced than oxidized glutathione.

[0082] Disulfide Bond Containing Bifunctional Molecules

[0083] Bifunctional molecules, possessing either homo orheterobifunctionality (commonly referred to as crosslinkers), are usedto connect two molecules together. The disulfide linkage (RSSR′) may beused within bifunctional molecules. The reversibility of disulfide bondformation makes them useful tools for the transient attachment of twomolecules. Physiologically, disulfides are reduced by glutathione.

[0084] A disulfide bond that is labile under physiological conditionsmeans: the disulfide bond is cleaved more rapidly than oxidizedglutathione or any disulfide constructed from thiols in which one of theconstituent thiols is more acidic, lower pKa, than glutathione or isactivated by intramolecular attack by a free thiol. Constituent in thiscase means the thiols that are bonded together in the disulfide bond.Cleavable means that a chemical bond between atoms is broken.

[0085] The present invention describes physiologically labile disulfidebond containing bifunctional molecules. The present invention is alsomeant to include constructs prepared from the bifunctional molecules,including polymers, peptides, proteins, nucleic acids, polymer nucleicacid complexes. Construct means any compound resulting from the chemicalreaction of at least one of the reactive centers of the bifunctionalmolecule resulting in new chemical bond other that that resulting fromhydrolysis of both reactive centers of the bifunctional molecule.Further chemical modification may occur after the formation of theconstruct. Crosslinking refers to the chemical attachment of two or moremolecules with a bifunctional reagent. A bifunctional reagent is amolecule with two reactive ends. The reactive ends can be identical asin a homobifunctional molecule, or different as in a heterobifunctionalmolecule.

[0086] Polymers

[0087] A polymer is a molecule built up by repetitive bonding togetherof smaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

[0088] To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer. “Most step-reactionpolymerizations are condensation processes and most chain-reactionpolymerizations are addition processes” (M. P. Stevens PolymerChemistry: An Introduction New York Oxford University Press 1990).Template polymerization can be used to form polymers from daughterpolymers.

[0089] Step Polymerization: In step polymerization, the polymerizationoccurs in a stepwise fashion. Polymer growth occurs by reaction betweenmonomers, oligomers and polymers. No initiator is needed since there isthe same reaction throughout and there is no termination step so thatthe end groups are still reactive. The polymerization rate decreases asthe functional groups are consumed.

[0090] Typically, step polymerization is done either of two differentways. One way, the monomer has both reactive functional groups (A and B)in the same molecule so that A—B yields —[A—B]—Or the other approach isto have two bifunctional monomers. A—A+B—B yields —[A—A—B—B]—Generally,these reactions can involve acylation or alkylation. Acylation isdefined as the introduction of an acyl group (—COR) onto a molecule.Alkylation is defined as the introduction of an alkyl group onto amolecule. If functional group A is an amine then B can be (but notrestricted to) an isothiocyanate, isocyanate, acyl azide,N-hydroxysuccinimide, sulfonyl chloride, aldehyde (includingformaldehyde and glutaraldehyde), ketone, epoxide, carbonate,imidoester, carboxylate activated with a carbodiimide, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, or acid halide,p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester,pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium, orcarbonyl dimethylaminopyridinium. In other terms when function A is anamine then function B can be acylating or alkylating agent or aminationagent.

[0091] If functional group A is a sulfhydryl then function B can be (butnot restricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoicacid{TNB} derivatives).

[0092] If functional group A is carboxylate then function B can be (butnot restricted to) a diazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

[0093] If functional group A is an hydroxyl then function B can be (butnot restricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used. Iffunctional group A is an aldehyde or ketone then function B can be (butnot restricted to) an hydrazine, hydrazide derivative, amine (to form aSchiff Base that may or may not be reduced by reducing agents such asNaCNBH3) or hydroxyl compound to form a ketal or acetal.

[0094] Yet another approach is to have one bifunctional monomer so thatA—A plus another agent yields —[A—A]—.

[0095] If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I2) or NaIO4 (sodiumperiodate), or oxygen (O2). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid {TNB} derivatives) can also be used tocatalyze disulfide bond formation. Functional group A or B in any of theabove examples could also be a photoreactive group such as aryl azide(including halogenated aryl azide), diazo , benzophenone, alkyne ordiazirine derivative.

[0096] Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groupsyield chemical bonds that are described as amide, amidine, disulfide,ethers, esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondary amine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone.

[0097] Chain Polymerization: In chain-reaction polymerization growth ofthe polymer occurs by successive addition of monomer units to limitednumber of growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.Monomers containing (but not limited to) vinyl, acrylate, methacrylate,acrylamide, methacrylamide groups can undergo chain reaction which canbe radical, anionic, or cationic. Chain polymerization can also beaccomplished by cycle or ring opening polymerization. Several differenttypes of free radical initiators could be used that include peroxides,hydroxy peroxides, and azo compounds such as2,2′-Azobis(-amidinopropane) dihydrochloride (AAP).

[0098] Types of Monomers

[0099] A wide variety of monomers can be used in the polymerizationprocesses. These include positive charged organic monomers such as aminesalts, imidine, guanidine, imine, hydroxylamine, hydrozyine, heterocycle(salts) like imidazole, pyridine, morpholine, pyrimidine, or pyrene. Theamines could be pH-sensitive in that the pKa of the amine is within thephysiologic range of 4 to 8. Specific amines include spermine,spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

[0100] Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties; carbohydrates,polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobicgroups.

[0101] Monomers can also be intercalating agents such as acridine,thiazole organge, or ethidium bromide. Monomers can also containchemical moieties that can be modified before or after thepolymerization including (but not limited to) amines (primary,secondary, and tertiary), amides, carboxylic acid, ester, hydroxyl,hydrazine, alkyl halide, aldehyde, and ketone.

[0102] Other Components of the Monomers and Polymers

[0103] The polymers have other groups that increase their utility. Thesegroups can be incorporated into monomers prior to polymer formation orattached to the polymer after its formation. These groups include:targeting groups, signals, reporter or marker molecules, spacers, stericstabilizers, chelators, polycations, polyanions, and polymers.

[0104] Targeting groups are used for targeting the polymer-nucleic acidcomplexes to specific cells or tissues. Examples of targeting agentsinclude agents that target to the asialoglycoprotein receptor by usingasiologlycoproteins or galactose residues. Proteins such as insulin,EGF, or transferrin can be used for targeting. Protein refers to amolecule made up of 2 or more amino acid residues connected one toanother by peptide bonds between the alpha-amino group and carboxylgroup of contiguous amino acid residues as in a polypeptide. The aminoacids may be naturally occurring or synthetic. Peptides that include theRGD sequence can be used to target many cells. Peptide refers to alinear series of amino acid residues connected to one another by peptidebonds between the alpha-amino group and carboxyl group of contiguousamino acid residues. Polypeptide includes proteins and peptides,modified proteins and peptides, and non-natural proteins and peptides.

[0105] Chemical groups that react with sulfhydryl or disulfide groups oncells can also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives.

[0106] Other targeting groups can be used to increase the delivery ofthe drug or nucleic acid to certain parts of the cell. For example,agents can be used to disrupt endosomes and a nuclear localizing signal(NLS) can be used to target the nucleus. A variety of ligands have beenused to target drugs and genes to cells and to specific cellularreceptors. The ligand may seek a target within the cell membrane, on thecell membrane or near a cell. Binding of ligands to receptors typicallyinitiates endocytosis. Ligands could also be used for DNA delivery thatbind to receptors that are not endocytosed. For example peptidescontaining RGD peptide sequence that bind integrin receptor could beused. In addition viral proteins could be used to bind the complex tocells. Lipids and steroids could be used to directly insert a complexinto cellular membranes. The polymers can also contain cleavable groupswithin themselves. When attached to the targeting group, cleavage leadsto reduce interaction between the complex and the receptor for thetargeting group. Cleavable groups include but are not restricted todisulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,acetals, ketals, enol ethers, enol esters, enamines and imines, acylhydrazones, and Schiff bases.

[0107] In a preferred embodiment, a chemical reaction can be used toattach a signal to a nucleic acid complex. The signal is defined in thisspecification as a molecule that modifies the nucleic acid complex, orbiologically active molecule, and can direct it to a cell location (suchas tissue cells) or location in a cell (such as the nucleus, orcytoplasm) either in culture or in a whole organism. By modifying thecellular or tissue location of the foreign gene, the expression of theforeign gene can be enhanced.

[0108] The signal can be a protein, peptide, lipid, steroid, sugar,carbohydrate, nucleic acid or synthetic compound. The signals enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

[0109] A certain subset of signals, termed transduction signals in thisapplication, transport themselves and attached molecules acrossmembranes (Schwarze and Dowdy Trends Pharm. Sci. 2000, 21, 45). Examplesof these transduction signals are derived from viral coat proteins suchas Tat from HIV and VP22 from herpes simplex virus, and atranscriptional factor from Drosophila, ANTP. The peptides Tat(Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), VP22(Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser-Arg-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro-Val-Glu),and ANTP(Arg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys) shareno sequence motif other than number of cationic (lysine and arginine)residues. In addition, reports of synthetic peptides possessing nohomology other than a propensity of cationic charge (net overallcationic charge) have also been shown to posses transduction activity(Service, R. F. Science 2000, 288, 28.)

[0110] Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

[0111] Signals that enhance release from intracellular compartments(releasing signals) can cause DNA release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin A1 and the ER-retaining signal (KDEL sequence),viral components such as influenza virus hemagglutinin subunit HA-2peptides and other types of amphipathic peptides. Cellular receptorsignals are any signal that enhances the association of the gene orparticle with a cell. This can be accomplished by either increasing thebinding of the gene to the cell surface and/or its association with anintracellular compartment, for example: ligands that enhance endocytosisby enhancing binding the cell surface. This includes agents that targetto the asialoglycoprotein receptor by using asiologlycoproteins orgalactose residues. Other proteins such as insulin, EGF, or transferrincan be used for targeting. Peptides that include the RGD sequence can beused to target many cells. Chemical groups that react with sulfhydryl ordisulfide groups on cells can also be used to target many types ofcells. Folate and other vitamins can also be used for targeting. Othertargeting groups include molecules that interact with membranes such aslipids fatty acids, cholesterol, dansyl compounds, and amphotericinderivatives. In addition viral proteins could be used to bind cells.

[0112] Reporter or marker molecules are compounds that can be easilydetected. Typically they are fluorescent compounds such as fluorescein,rhodamine, Texas red, cy 5, cy 3 or dansyl compounds. They can bemolecules that can be detected by UV or visible spectroscopy or byantibody interactions or by electron spin resonance. Biotin is anotherreporter molecule that can be detected by labeled avidin. Biotin couldalso be used to attach targeting groups.

[0113] A spacer is any linker known to those skilled in the art toenable one to join one moiety to another moiety. The moieties can behydrophilic or hydrophobic. Preferred spacer groups include, but are notlimited to C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, C6-C18 aralkyl,C6-C 18 aralkenyl, C6-C 18 aralkynyl, ester, ether, ketone, alcohol,polyol, amide, amine, polyglycol, polyamine, thiol, thio ether,thioester, phosphorous containing, and heterocyclic.

[0114] A Steric stabilizer is a long chain hydrophilic group thatprevents aggregation of final polymer by sterically hindering particleto particle electrostatic interactions. Examples include: alkyl groups,PEG chains, polysaccharides, hydrogen molecules, alkyl amines.Electrostatic interactions are the non-covalent association of two ormore substances due to attractive forces between positive and negativecharges.

[0115] A polycation is a polymer containing a net positive charge, forexample poly-L-lysine hydrobromide. The polycation can contain monomerunits that are charge positive, charge neutral, or charge negative,however, the net charge of the polymer must be positive. A polycationalso can mean a non-polymeric molecule that contains two or morepositive charges. A polyanion is a polymer containing a net negativecharge, for example polyglutamic acid. The polyanion can contain monomerunits that are charge negative, charge neutral, or charge positive,however, the net charge on the polymer must be negative. A polyanion canalso mean a non-polymeric molecule that contains two or more negativecharges. The term polyion includes polycation, polyanion, zwitterionicpolymers, and neutral polymers. The term zwitterionic refers to theproduct (salt) of the reaction between an acidic group and a basic groupthat are part of the same molecule. Salts are ionic compounds thatdissociate into cations and anions when dissolved in solution. Saltsincrease the ionic strength of a solution, and consequently decreaseinteractions between nucleic acids with other cations.

[0116] A chelator is a polydentate ligand, a molecule that can occupymore than one site in the coordination sphere of an ion, particularly ametal ion, primary amine, or single proton. Examples of chelatorsinclude crown ethers, cryptates, and non-cyclic polydentate molecules. Acrown ether is a cyclic polyether containing (—X—(CR1-2)n)m units, wheren=1-3 and m=3-8. The X and CR1-2 moieties can be substituted, or at adifferent oxidation states. X can be oxygen, nitrogen, or sulfur,carbon, phosphorous or any combination thereof. R can be H, C, O, S, N,P. A subset of crown ethers described as a cryptate contain a second(—X—(CR1-2)n)z strand where z=3-8. The beginning X atom of the strand isan X atom in the (—X—(CR1-2)n)m unit, and the terminal CH2 of the newstrand is bonded to a second X atom in the (—X—(CR1-2)n)m unit.Non-cyclic polydentate molecules containing (—X—(CR1-2)n)m unit(s),where n=1-4 and m=1-8. The X and CR1-2 moieties can be substituted, orat a different oxidation states. X can be oxygen, nitrogen, or sulfur,carbon, phosphorous or any combination thereof. A polychelator is apolymer associated with a plurality of chelators by an ionic or covalentbond and can include a spacer. The polymer can be cationic, anionic,zwitterionic, neutral, or contain any combination of cationic, anionic,zwitterionic, or neutral groups with a net charge being cationic,anionic or neutral, and may contain steric stabilizers, peptides,proteins, signals, or amphipathic compound for the formation ofmicellar, reverse micellar, or unilamellar structures. Preferably theamphipathic compound can have a hydrophilic segment that is cationic,anionic, or zwitterionic, and can contain polymerizable groups, and ahydrophobic segment that can contain a polymerizable group.

[0117] The present invention provides for the transfer ofpolynucleotides, and biologically active compounds into parenchymalcells within tissues in situ and in vivo, utilizing disulfide bonds thatcan be cleaved under physialogicval condidtions, and deliveredintravasculary (U.S. patent application Ser. No. 08/571,536),intrarterially, intravenous, orally, intraduodenaly, via the jejunum (orileum or colon), rectally, transdermally, subcutaneously,intramuscularly, intraperitoneally, intraparenterally, via directinjections into tissues such as the liver, lung, heart, muscle, spleen,pancreas, brain (including intraventricular), spinal cord, ganglion,lymph nodes, lymphatic system, adipose tissues, thryoid tissue, adrenalglands, kidneys, prostate, blood cells, bone marrow cells, cancer cells,tumors, eye retina, via the bile duct, or via mucosal membranes such asin the mouth, nose, throat, vagina or rectum or into ducts of thesalivary or other exocrine glands.

[0118] “Delivered” means that the polynucleotide becomes associated withthe cell. The polynucleotide can be on the membrane of the cell orinside the cytoplasm, nucleus, or other organelle of the cell. Theprocess of delivering a polynucleotide to a cell has been commonlytermed “transfection” or the process of “transfecting” and also it hasbeen termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The polynucleotides orgenetic material being delivered are generally mixed with transfectionreagents prior to delivery.

[0119] A biologically active compound is a compound having the potentialto react with biological components. More particularly, biologicallyactive compounds utilized in this specification are designed to changethe natural processes associated with a living cell. For purposes ofthis specification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. In this specification, the cellular production of, orinhibition of a material, such as a protein, caused by a human assistinga molecule to an in vivo cell is an example of a delivered biologicallyactive compound. Pharmaceuticals, proteins, peptides, polypeptides,hormones, cytokines, antigens, viruses, oligonucleotides, and nucleicacids are examples of biologically active compounds. Bioactive compoundsmay be used interchangeably with biologically active compound forpurposes of this application.

[0120] The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nuclei acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). DNA may be in the form of anti-sense, plasmid DNA, parts ofa plasmid DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is apolynucleotide that interferes with the function of DNA and/or RNA. Thismay result in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones and bases. These include PNAs (peptide nucleic acids),phosphothionates, and other variants of the phosphate backbone of nativenucleic acids.

[0121] In addition, DNA and RNA may be single, double, triple, orquadruple stranded. “Expression cassette” refers to a natural orrecombinantly produced polynucleotide molecule which is capable ofexpressing protein(s). A DNA expression cassette typically includes apromoter (allowing transcription initiation), and a sequence encodingone or more proteins. Optionally, the expression cassette may includetrancriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include translationtermination signals, a polyadenosine sequence, internal ribosome entrysites (IRES), and non-coding sequences.

[0122] The term “naked polynucleotides” indicates that thepolynucleotides are not associated with a transfection reagent or otherdelivery vehicle that is required for the polynucleotide to be deliveredto the cardiac muscle cell. A “transfection reagent” or “deliveryvehicle” is a compound or compounds used in the prior art that bind(s)to or complex(es) with oligonucleotides or polynucleotides, and mediatestheir entry into cells. The transfection reagent also mediates thebinding and internalization of polynucleotides into cells. Examples oftransfection reagents include cationic liposomes and lipids, polyamines,calcium phosphate precipitates, histone proteins, polyethylenimine, andpolylysine complexes (polyethylenimine and polylysine are both toxic).Typically, the transfection reagent has a net positive charge that bindsto the polynucleotide's negative charge. The transfection reagentmediates binding of polynucleotides to cell via its positive charge(that binds to the cell membrane's negative charge) or via ligands thatbind to receptors in the cell. For example, cationic liposomes orpolylysine complexes have net positive charges that enable them to bindto DNA or RNA. Other delivery vehicles are also used, in the prior art,to transfer genes into cells. These include complexing thepolynucleotides on particles that are then accelerated into the cell.This is termed “biolistic” or “gun” techniques.

[0123] Ionic (electrostatic) interactions are the non-covalentassociation of two or more substances due to attractive forces betweenpositive and negative charges, or partial positive and partial negativecharges.

[0124] Condensed Nucleic Acids: A method of condensing a polymer isdefined as decreasing its linear length, also called compacting.Condensing a polymer also means decreasing the volume that the polymeroccupies. An example of condensing nucleic acid is the condensation ofDNA that occurs in cells. The DNA from a human cell is approximately onemeter in length but is condensed to fit in a cell nucleus that has adiameter of approximately 10 microns. The cells condense (or compacts)DNA by a series of packaging mechanisms involving the histones and otherchromosomal proteins to form nucleosomes and chromatin. The DNA withinthese structures is rendered partially resistant to nuclease DNase)action. The process of condensing polymers can be used for deliveringthem into cells of an organism. A delivered polymer can stay within thecytoplasm or nucleus apart from the endogenous genetic material.Alternatively, the polymer could recombine (become a part of) theendogenous genetic material. For example, DNA can insert intochromosomal DNA by either homologous or non-homologous recombination.

[0125] Intravascular: An intravascular route of administration enables apolymer or polynucleotide to be delivered to cells more evenlydistributed and more efficiently expressed than direct injections.Intravascular herein means within a tubular structure called a vesselthat is connected to a tissue or organ within the body. Within thecavity of the tubular structure, a bodily fluid flows to or from thebody part. Examples of bodily fluid include blood, lymphatic fluid, orbile. Examples of vessels include arteries, arterioles, capillaries,venules, sinusoids, veins, lymphatics, and bile ducts. The intravascularroute includes delivery through the blood vessels such as an artery or avein. An administration route involving the mucosal membranes is meantto include nasal, bronchial, inhalation into the lungs, or via the eyes.

[0126] Buffers are made from a weak acid or weak base and their salts.Buffer solutions resist changes in pH when additional acid or base isadded to the solution. Biological, chemical, or biochemical reactionsinvolve the formation or cleavage of ionic and/or covalent bonds.Biomolecule refers to peptides, polypeptides, proteins, enzymes,polynucleotides, oligonucleotides, viruses, antigens, carbohydrates (andconjugates), lipids, and saccharides. Enzymes are proteins evolved bythe cells of living organisms for the specific function of catalyzingchemical reactions. A chemical reaction is defined as the formation orcleavage of covalent or ionic bonds. As a result of the chemicalreaction a polymer can be formed. A polymer is defined as a compoundcontaining more than two monomers. A monomer is a compound that can beattached to itself or another monomer and thus a form a polymer.

[0127] Transdermal refers to application to mammal skin in which drugdelivery occurs by crossing the dermal layer.

[0128] Hydrocarbon means containing carbon and hydrogen atoms; andhalohydrocarbon means containing carbon, halogen (F, Cl, Br, I), andhydrogen atoms.

[0129] Alkyl means containing sp³ hybridized carbon atoms; alkenyl meanscontaining two or more sp² hybridized carbon atoms; aklkynyl meanscontaining two or more sp hybridized carbon atoms; aralkyl meanscontaining one or more aromatic ring(s) in addition containing sp³hybridized carbon atoms; aralkenyl means containing one or more aromaticring(s) in addition to containing two or more sp² hybridized carbonatoms; aralkynyl means containing one or more aromatic ring(s) inaddition to containing two or more sp hybridized carbon atoms; steroidincludes natural and unnatural steroids and steroid derivatives.

[0130] A steroid derivative means a sterol, a sterol in which thehydroxyl moity has been modified (for example, acylated), or a steroidhormone, or an analog thereof.

[0131] Carbohydrates include natural and unnatural sugars (for exampleglucose), and sugar derivatives (a sugar derivative means a system inwhich one or more of the hydroxyl groups on the sugar moiety has beenmodified (for example acylated), or a system in which one or more of thehydroxyl groups is not present).

[0132] Polyoxyethylene means a polymer having two to six (n=2-3000)ethylene oxide units (—(CH₂CH₂O)_(n)—) or a derivative thereof.

[0133] R is meant to be any compatible group, for example hydrogen,alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, and caninclude heteroatoms (N, O, S), and carbonyl groups.

[0134] A compound is a material made up of two or more elements.

[0135] Electron withdrawing group is any chemical group or atom composedof electronegative atom(s), that is atoms that tend to attractelectrons. Resonance stabilization is the ability to distribute chargeon multiple atoms through pi bonds. The inductive effective, in amolecule, is a shift of electron density due to the polarization of abond by a nearby electronegative or electropositive atom.

[0136] Steric hindrance, or sterics, is the prevention or retardation ofa chemical reaction because of neighboring groups on the same molecule.

[0137] An activated carboxylate is a carboxylic acid derivative thatreacts with nucleophiles to form a new covalent bond. Nucleophilesinclude nitrogen, oxygen and sulfur-containing compounds to produceureas, amides, carbonates, esters, and thioesters. The carboxylic acidmay be activated by various agents including carbodiimides, carbonates,phosphoniums, uroniums to produce activated carboxylates acyl ureas,acylphosphonates, and carbonates. Activation of carboxylic acid may beused in conjunction with hydroxy and amine-containing compounds toproduce activated carboxylates N-hydroxysuccinimide esters,hydroxybenzotriazole esters,N-hydroxy-5-norbomene-endo-2,3-dicarboximide esters, p-nitrophenylesters, pentafluorophenyl esters, 4-dimethylaminopyridinium amides, andacyl imidazoles.

[0138] A nucleophile is a species possessing one or more electron-richsites, such as an unshared pair of electrons, the negative end of apolar bond, or pi electrons.

EXAMPLES Example 1 Synthesis of Cysteine-terminal Tat Peptide (Tat-Cys).

[0139] Peptide syntheses were performed using standard solid phasepeptide techniques using FMOC chemistry. A cysteine was added to theamino terminus of Tat to allow for conjugation through the thiol groupto make the peptide Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys(Tat-Cys).

Example 2 Synthesis of Noncleavably Linked (Irreversible Covalent)Tat-Cys and Fluorescein through a Thioether Bond.

[0140] To a solution of succinimidyl-4-(N-maleimidomethyl)cyclohexane-carboxylate (SMCC from Pierce) 1.0 mg in 0.1 mLdimethylformamide was added 1.2 mg (1 eq) of4′-(aminomethyl)fluorescein. After two hours, this solution was added toa 1 mL aqueous solution of 8.4 mg Tat-Cys (1 eq). The solution wasbuffered to pH 8 by the addition of potassium carbonate. This solutionwas used for transport studies without further purification.

Example 3 Synthesis of Unactivated (Non-labile) Disulfide LinkedLissamine Dimer (Dilissamine Cystamine)

[0141] To a solution of cystamine dihydrochloride (10 mg) in water (1mL) was added diisopropylethylamine (15 μL, 2 eq). To this was addedlissamine chloride (Rhodamine B sulfonyl chloride, Molecular Probes) 77mg (3 eq) in 5 mL of methanol. The solution was stirred for 1 hour andthen chromatographed by reverse-phase HPLC using an Aquasil C-18 columnusing a gradient from 100% 0.1% trifluoroacetic acid in water to 100%0.1% triflouroacetic acid in acetonitrile. The fraction containing theproduct was determined by mass spectroscopy. The molecular weight ofcompound is 1234, which was detected in positive ion mode. Theconcentration of the product-containing fraction was determined by theabsorbance of the solution at 588 nm (ε=88,000 M⁻¹ cm⁻¹).

Example 4 Attachment of Lissamine to Tat-Cys by an Unactivated Disulfide

[0142] To a solution of Tat-Cys (100 μg) in 100 μL water was addeddilissamine cystamine (41 μg, 1 eq). The pH of the solution was adjustedto 7-8 by the addition of potassium carbonate.

Example 5 Synthesis of Activated (Labile) Disulfide-containing LissamineAdduct (Lissamine 4-aminophenyl Disulfide)

[0143] To a solution of 10 mg of lissamine chloride (Rhodamine Bsulfonyl chloride, Molecular Probes) in 0.2 mL dimethylformamide wasadded over five minutes ten 10 μL aliquots of 4-aminophenyl disulfide (2mg, 0.5 eq) and diisopropylethylamine (3 μL, 1 eq). Two hours afterfinal addition of disulfide the solution was diluted into 2 mL ofacetonitrile and chromatographed by reverse-phase HPLC using an AquasilC-18 column applying a gradient from 20% acetonitrile and 80% watercontaining 0.1% trifluoroacetic acid to 100% 0.1% triflouroacetic acidin acetonitrile. We were unable to isolate the lissamine dimer, but wereable to isolate the product of monoaddition. The fraction containing themonoaddition product was determined by mass spectroscopy. The molecularweight of compound is 789, which was detected in positive ion mode. Theconcentration of the product-containing fraction was determined by theabsorbance of the solution at 588 nm (ε=88,000 M⁻¹ cm⁻¹).

Example 6 Attachment of Lissamine to Tat-Cys by an Activated Disulfide

[0144] To a solution of Tat-Cys (100 μg) in 100 μL water was addedLissamine 4-aminophenyl disulfide (26 μg, 1 eq). The pH of the solutionwas adjusted to 7-8 by the addition of potassium carbonate.

Example 7 Synthesis of Activated (Labile) Disulfide Fluorescein Dimer(Difluorescein 4-aminophenyl Disulfide)

[0145] To a solution of 20 mg of fluorescein isothiocyanate in 0.5 mLdimethylformamide was added 4-aminophenyl disulfide (4 mg, 0.33 eq) in100 μL dimethylformamide and diisopropylethylamine (3 μL, 0.33 eq).After two hours, the solution was diluted into 2 mL of water that wasbrought to pH 8 with potassium carbonate. This aqueous solution wasfiltered and chromatographed by reverse-phase HPLC using an Aquasil C-18column applying a gradient from 100% water containing 0.1%trifluoroacetic acid to 100% 0.1% triflouroacetic acid in acetonitrile.The fraction containing the product was determined by mass spectroscopy.The molecular weight of compound is 1025, which was detected in negativeion mode. The concentration of the product-containing fraction wasdetermined by the absorbance of the solution at 494 nm (ε=75,000 M⁻¹cm⁻¹).

Example 8 Measurement of the Reduction of Unactivated (Non-labile)Disulfide (Dilissamine Cystamine).

[0146] To a solution containing 0.44 μM dilissamine cystamine and 100 mMsodium phosphate pH 7.5 was added glutathione to a concentration of 250μM. The solution was irradiated with 555 nm light and the fluorescenceof the solution was measured at 585 nm. The amount of time required toreach half maximum fluorescence was 2000-2400 sec.

Example 9 Measurement of the Reduction of Activated (Labile) Disulfide(Difluorescein 4-aminophenyl Disulfide).

[0147] To a solution containing 0.44 μM fluorescein 4-aminophenyldisulfide and 100 mM sodium phosphate pH 7.5 was added glutathione to aconcentration of 250 μM. The solution was irradiated with 495 nm lightand the fluorescence of the solution was measured at 520 nm. The amountof time required to reach half maximum fluorescence was 30-50 sec.

Example 10 Analysis of Delivery to Cells by TAT Peptide.

[0148] Grow HeLa cells on glass coverslips by incubating at 4° C. inDelbecco's Modified Eagle's Media (DMEM) supplemented with 50 μg TATpeptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external TAT-fluorophore. Removethe media and then either process cells for fluorescence microscopy orincubate three more hours at 4° C. with DMEM with media changes everyhour (chase). The cells that are chased are then processed forfluorescence microscopy. Cells processed for fluorescence microscopy arewashed 3× in phosphate-buffered saline (PBS), fixed in PBS+4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides.

[0149] The presence of fluorophore was detected by confocal microscopy(Zeiss LSM 510). In the case of irreversible covalent thioether linkagebetween TAT and fluorophore, fluorescence was detected inside of thecell after the initial two hour incubation. Subsequent incubation of thecells with fluorophore-free media (chase) resulted in cells with nointernalized fluorophore. Similarly, TAT-fluorophore adducts linkedthrough an unactivated disulfide cystamine bond also had initialinternalization that disappeared upon incubation with chase solutions.For the activated disulfide 4-aminophenyl disulfide, fluorescence wasdetected inside of the cell after the initial two hour incubation. Incontrast to the other attachments between flourophore and TAT, a chaseof the fluorophore with fluorophore-free media did not show a reductionin the amount of internalized fluorophore.

Example 11 Synthesis of 5,5′-Dithiobis(2-nitrobenzoate)propionitrile

[0150] 5,5′-dithiobis(2-nitrobenzoic acid) (500 mg, 1.26 mmol, AldrichChemical Company) was taken up in 4.0 mL dioxane.Dicylohexylcarbodiimide (540 mg, 2.6 mmol, Aldrich Chemical Company) and3-hydroxypropionitrile (240 μL, 188 mg, 2.60 mmol, Aldrich ChemicalCompany) were added. The reaction mixture was stirred overnight at roomtemperature. The precipitate was removed by centrifugation, and thesolvent concentrated under reduced pressure. The residue was washed withsaturated sodium bicarbonate, water, and brine; and dried over magnesiumsulfate. Solvent removal (aspirator) yielded 696 mg yellow/orange foam.The residue was purified using normal phase HPLC (Alltech econosil,250×22 nm), flow rate=9.0 mL/min, mobile phase=1% ethanol in chloroform,retention time=13 min. Removal of solvent (aspirator) afforded 233 mg(36.8%) of 5,5′-dithiobis(2-nitrobenzoate)propionitrile as a yellow oil.TLC (silica: 5% methanol in chloroform; Rf=0.51). H¹NMR ∂ 8.05 (d, 4 H),7.75 (m, 4H), 4.55 (t, 4H), 2.85 (t, 4H).

Example 12 Synthesis of Dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl

[0151] 5,5′-Dithiobis(2-nitrobenzoate)propionitrile (113 mg, 0.226 mmol)was taken up in 500. μL anhydrous chloroform. Anhydrous methanol (20.0μL, 0.494 mmol, Aldrich Chemical Company) was added. The resultingsolution was cooled to 0° C. on an ice bath, and HCl gas was bubbledthrough the solution for a period of 10 minutes. The resulting solutionwas placed in a −20° C. freezer for a period of 48 hours. During thistime a yellow oil formed. The oil was washed thoroughly with chloroformand dried under vacuum to afford 137 mg (95.8%) of dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl as a yellow foam.

Example 13 Polymerization of N-(2-Aminoethyl)-1,3-propanediamine andDimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl on a DNATemplate.

[0152] Procedure:

[0153] Template polymerization was carried out in 25 mM HEPES buffer, pH8.0. N-(2-Aminoethyl)-1,3-propanediamine (48 μg, 0.3 mM, AldrichChemical Company) was added to a 0.5 mL solution of pCIluc DNA (25 mg,0.075 mM in phosphate, 2.6 μg/μL pCIluc; prepared according to Danko,I., Williams, P., Herweijer, H. et al. Hum. Mol. Genetics (1997) inpress). Dimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl(500 μg, 0.78 mM) was added, and the solution was vortexed. The reactionwas incubated at room temperature for one hour. A fine yellowprecipitate was observed to form during the incubation period. Thereaction was centrifuged to remove the precipitate. A portion of thereaction (10 μL) was reduced with 10 mM dithiothreitol (10 μL) to breakthe disulfide bonds forming the polymer. Portions (0.5 μg) of the intactpolymer and the reduced polymer were analyzed on a 1% agarose gel.

Example 14 Formation of DNA/Poly-L-Lysine/Dimethyl5,5′-Dithiobis(2-nitrobenzoate) propionimidate -2 HCl Complexes

[0154] pDNA/Poly-L-lysine hydrobromide complexes were prepared bycombining plasmid DNA (25 μg) with Poly-L-lysine hydrobromide (95 μg, MW35 kDa, Aldrich Chemical Company) in 0.5 mL 25 mM Hepes buffer pH 8.0,and the solution was vortexed to mix. The resulting solution was dividedinto 3 portions. One portion was incubated at room temperature for 2hrs. To the second portion was added dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl (472 mg, 1.5 mmol),the solution was mixed, and incubated at room temperature for 2 hrs. Tothe third sample was added dimethyl 3,3′-dithiobispropionimidate (1.1mg, 1.5 mmol), the solution was mixed, and incubated at room temperaturefor 2 hrs. After 2 hrs. the samples were then centrifuged at 12000 rpmfor five minutes.

[0155] Ninety degree light scattering measurements were performed(Shimadzo RF-1501 Fluorescence Spectrophotometer). The wavelengthsetting was 700 nm for both the incident beam and detection ofscattering light. The slits for both beams were fixed at 10 nm. Theparticle size of the resulting complex was determined by lightscattering (Brookhaven ZetaPlus Particle Sizer). After determining theinitial intensity of scattered light, 15 μL 5 M NaCl solution was addedto the complexes while the intensity of scattered light was monitored.

[0156] The addition of salt to the non-caged particles led to animmediate increase in the turbidity of the solution indicatingaggregation. The non-caged sample also became visibly cloudy. Theaddition of salt to the particles caged using dimethyl3,3′-dithiobispropionimidate led to an increase in turbidity ofapproximately 33%. The addition of salt to the dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl caged complexes leadto no visible rise in turbidity. The particle size of the dimethyl5,5′-dithiobis(2-nitrobenzoate) propionimidate-2 HCl caged particles wasdetermined (Brookhaven Zeta Plus Particle Sizer) in 150 mM NaCl(physiological concentration). The mean particle diameter was found tobe 89.7 nm, 67% of the total number of particles were under 100 nm insize.

[0157] The example indicates that dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl caged DNA. Theparticles formed are stable in physiological salt, and are under 100 nmin size.

Example 15 Demonstration of Reducibility of Disulfide Bond in vitro.

[0158] pDNA (pCI Luc)/polyethyleneimine (25 kDa, Aldrich ChemicalCompany)/dimethyl 3,3′-dithiobispropionimidate andpDNA/polyethyleneimine/dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl complexes wereprepared in 25 mM HEPES buffer pH 8.0. All complexes were prepared atpDNA/polyethyleneimine ratios of 1/3. Dimethyl3,3′-dithiobispropionimidate and dimethyl5,5′-dithiobis(2-nitrobenzoate) propionimidate-2 HCl were added at thefollowing ratios: 0, 3, 6, 12, and 25. Complexes were incubated 0.5 hourat room temperature, and centrifuged 5 minutes at 12,000 rpm prior totransfection. Transfections were carried out in 35 mm wells. At the timeof transfection, HepG2 monolayers, at approximately 50% confluency, werewashed once with PBS (phosphate buffered saline), and subsequentlystored in serum-free media (Opti-MEM, Gibco BRL). The complexes werediluted in Opti-MEM and added by drops, 5.0 μg DNA/well, to the cells.After a 4 hour incubation period at 37° C., the media containing thecomplexes was aspirated from the cells, and replaced with completegrowth media, DMEM with 10% fetal bovine serum (Sigma). After anadditional incubation of 42 hours, the cells were harvested and thelysate was assayed for luciferase expression (Wolff, J. A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Feigner, P. L.Direct gene transfer into mouse muscle in vivo. Science,1465-1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used.

[0159] pDNA/polyethyleneimine/methyl 3,3′-dithiobispropionimidate andpDNA/polyethyleneimine/dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl particles weretransfected into Hep G2 cells. pDNA/polyethyleneimine complexes werealso transfected as a control. The cell lysates were then analyzed forthe expression of luciferin. The results show that while the dimethyl3,3′-dithiobispropionimidate complexes gave expression results belowbaseline (<200 RLU), the dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2HCl/pDNA/polyethyleneimine complexes gave levels of expression that wereas high as 120,000 RLU.

[0160] The physiologically labile disulfide bonds present in thedimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl complexescan be reduced by cultured cells, while the disulfide bonds present inthe dimethyl 3,3′-dithiobispropionimidate complexes cannot.

Example 16 Synthesis of 5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate]

[0161] 5,5′-dithiobis-(2-nitrobenzoic acid) (500 mg, 1.26 mmol, AldrichChemical Company) and 3-bromopropanol (368 mg, 2.65 mmol, AldrichChemical Company) were taken up in 7.0 mL THF. Dicyclohexylcarbodiimide(545 mg, 2.65 mmol, Aldrich Chemical Company) was added, and thereaction mixture was stirred overnight at ambient temperature. Theprecipitate was removed by filtration, and the solution was concentratedunder reduced pressure to afford 430 mg (54%) of5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate] as a yellow oil.

Example 17 Synthesis of5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate]

[0162] 5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate] was taken up in2.0 mL THF, and 3-dimethylaminopropionitrile (193 mg, 1.96 mmol, AldrichChemical Company) was added. After 3 days at ambient temperature, thesalt was precipitated from solution with Et₂O, and purified by reversephase HPLC (C-18 Aquasil 200×20 mm) using a gradient from 20 to 80%methanol over 20 minutes (elution at 15 minutes). The solvent wasremoved under reduced pressure to afford 15.2 mg (3%)5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate]. H¹-NMR (CD₃OD) ∂ 8.4-8.6 (m, 6H), 5.0 (t, 4H), 4.35 (t, 4H), 4.1 (m, 4H), 2.85 (m, 4H), 3.75 (m, 16H). Synthesis ofDimethyl 5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]-hydrochloride

Example 18 Synthesis of5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]

[0163] 5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate] (15.2 mg, 0.018 mmol) was taken up in 1 mL ofmethanol. The solution was saturated with HCl at 0° C. The resultingsolution was held at −20° C. for 1 week. Et₂O was added and theprecipitate collected by filtration to afford 8.3 mg (47%) of dimethyl5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]-hydrochloride.

Example 19 Synthesis of N,N′-Bis(t-BOC)-L-cystine

[0164] To a solution of L-cystine (1 gm, 4.2 mmol, Aldrich ChemicalCompany) in acetone (10 mL) and water (10 mL) was added2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (2.5 gm, 10 mmol,Aldrich Chemical Company) and triethylamine (1.4 mL, 10 mmol, AldrichChemical Company). The reaction was allowed to stir overnight at roomtemperature. The water and acetone was then by rotary evaporationresulting in a yellow solid. The diBOC compound was then isolated byflash chromatography on silica gel eluting with ethyl acetate 0.1%acetic acid.

Example 20 Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazineCopolymer

[0165] To a solution of N,N′-Bis(t-BOC)-L-cystine (85 mg, 0.15 mmol) inethyl acetate (20 mL) was added N,N′-dicyclohexylcarbodiimide (108 mg,0.5 mmol) and N-hydroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, thesolution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (54 μL, 0.25 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h. The ethylacetate was then removed by rotary evaporation and the resulting solidwas dissolved in trifluoroacetic acid (9.5 mL), water (0.5 mL) andtriisopropylsilane (0.5 mL). After 2 h, the trifluoroacetic acid wasremoved by rotary evaporation and the aqueous solution was dialyzed in a15,000 MW cutoff tubing against water (2×2 l) for 24 h. The solution wasthen removed from dialysis tubing, filtered through 5 μM nylon syringefilter and then dried by lyophilization to yield 30 mg of polymer.

Example 21 Synthesis of Guanidino-L-cystine

[0166] To a solution of cystine (1 gm, 4.2 mmol) in ammonium hydroxide(10 mL) in a screw-capped vial was added O-methylisourea hydrogensulfate (1.8 gm, 10 mmol). The vial was sealed and heated to 60° C. for16 h. The solution was then cooled and the ammonium hydroxide wasremoved by rotary evaporation. The solid was then dissolved in water (20mL), filtered through a cotton plug. The product was then isolated byion exchange chromatography using Bio-Rex 70 resin and eluting withhydrochloric acid (100 mM).

Example 22 Synthesis ofGuanidino-L-cystine-L-1,4-bis(3-aminopropyl)piperazine Copolymer

[0167] To a solution of guanidino-L-cystine (64 mg, 0.2 mmol) in water(10 mL) was slowly added N,N′-dicyclohexylcarbodiimide (82 mg, 0.4 mmol)and N-hyroxysuccinimide (46 mg, 0.4 mmol) in dioxane (5 mL). After 16hr, the solution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (40 μL, 0.2 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h and then theaqueous solution was dialyzed in a 15,000 MW cutoff tubing against water(2×2 l) for 24 h. The solution was then removed from dialysis tubing,filtered through 5 μM nylon syringe filter and then dried bylyophilization to yield 5 mg of polymer.

Example 23 The Particle Size ofpDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer andDNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine CopolymerComplexes.

[0168] To a solution of pDNA (10 μg/mL) in 0.5 mL 25 mM HEPES buffer pH7.5 was added 10 μg/mL L-cystine-1,4-bis(3-aminopropyl)piperazinecopolymer or guanidino-L-cystine1,4-bis(3-aminopropyl)piperazinecopolymer. The size of the complexes between DNA and the polymers weremeasured. For both polymers, the size of the particles wereapproximately 60 nm.

Example 24 Condensation of DNA withL-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer and Decondensationof DNA upon Addition of Glutathione

[0169] Fluorescein labeled DNA was used for the determination of DNAcondensation in complexes withL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. pDNA was modifiedto a level of 1 fluorescein per 100 bases using Mirus' LabelI™Fluorescein kit. The fluorescence was determined using a fluorescencespectrophotometer (Shimadzu RF-1501 spectrofluorometer) at an excitationwavelength of 495 nm and an emission wavelength of 530 nm. (Trubetskoy,V. S., Slattum, P. M., Hagstrom, J. E., Wolff, J. A., Budker, V. G.,“Quantitative Assessment of DNA Condensation,” Anal. Biochem (1999)incorporated by reference).

[0170] The intensity of the fluorescence of the fluorescein-labeled DNA(10 μg/mL) in 0.5 mL of 25 mM HEPES buffer pH 7.5 was 300 units. Uponaddition of 10 μg/mL of L-cystine-1,4-bis(3-aminopropyl)piperazinecopolymer, the intensity decreased to 100 units. To this DNA-polycationsample was added 1 mM glutathione and the intensity of the fluorescencewas measured. An increase in intensity was measured to the levelobserved for the DNA sample alone. The half life of this increase influorescence was 8 minutes.

[0171] The experiment indicates that DNA complexes withphysiologically-labile disulfide-containing polymers are cleavable inthe presence of the biological reductant glutathione.

Example 25 Mouse Tail Vein Injection ofDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer andDNA-guanidino-L-cystinel,4-bis(3-aminopropyl)piperazine CopolymerComplexes

[0172] Plasmid delivery in the tail vein of ICR mice was performed asdescribed. To PCILuc DNA (50 μg) in 2.5 mL H₂O was added eitherL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer,guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer, orpoly-L-lysine (34,000 MW, Sigma Chemical Company) (50 μg). The sampleswere then injected into the tail vein of mice using a 30 gauge, 0.5 inchneedle. One day after injection, the animal was sacrificed, and aluciferase assay was conducted. Polycation ng/liver poly-L-lysine 6.2L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 439guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer 487

[0173] The experiment indicates that DNA complexes with thephysiologically-labile disulfide-containing polymers are capable ofbeing broken, thereby allowing the luciferase gene to be expressed.

Example 26 Rat Intramuscle injection ofDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer andDNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymercomplexes.

[0174] Plasmid delivery intro rat leg was performed as described (Wolff,J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465-1468,1990.). To pCILuc DNA (100 μg/mL, 2.5 mL) was addedL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer orguanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer (100μg/mL) and then injected into the leg muscles of a rat. After 7 days,the animal was sacrificed and a luciferase assay was conducted. amountluciferase DNA complex (ng) per leg no polycation 3.3L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 4.5guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine 6.5 copolymer

[0175] The experiment indicates that DNA complexes with thephysiologically-labile disulfide-containing polymers are capable ofbeing broken, thereby allowing the luciferase gene to be expressed.

Example 27 Injection of DNA-L-cystine-1,4-bis(3-aminopropyl)piperazineCopolymer Complex and pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complex and pDNA (pCILuc)/5,5′-dithiobis(2-nitrobenzoicAcid)-1,4-bis(3-aminopropyl)piperazine—Folate Copolymer Complexes intothe Intestinal Lumen of Mice.

[0176] Intestinal cells were transfected by injecting pDNA solutionsinto the mesenteric vasculature. A 3-cm section of the small intestineswas clamped, blocking both vascular inflow and outflow. A volume of 250μl containing 50 μg pCILuc and 50 μg poly(ethylenimine) (AldrichChemical Co. MW 25,000 MW), L-cystine-1,4-bis(3-aminopropyl)piperazinecopolymer, pDNA (pCI Luc)/5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine copolymer, and pDNA (pCILuc)/5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine-folate copolymer complexes wereinjected into the intestinal lumen of mice. After 3 minutes, the clampswere removed. One day after DNA delivery, the mice were sacrificed, theinjected section of the intestines was excised, cut in 3 cm sections andassayed for luciferase expression. Different areas of the intestineswere targeted (duodenum, jejunum, ileum). Amount luciferase (pg) ComplexDuodenum jejunum ileum DNA-poly(ethylenimine) 0.5 3.0 1.7DNA-L-cystine-1,4-bis 6.2 3.7 2.8 (3-aminopropyl)piperazine copolymerpDNA (pCI Luc)/5,5′-dithiobis(2-nitro- 42 20 226 benzoicacid)-1,4-bis(3-amino- propyl)piperazine copolymer pDNA (pCILuc)/5,5′-dithiobis(2-nitro- 36 1.9  51 benzoic acid)-1,4-bis(3-amino-propyl)piperazine-folate copolymer

[0177] The experiment indicates that DNA complexes with labiledisulfide-containing polymers are capable of being broken, therebyallowing the luciferase gene to be expressed.

Example 28 Synthesis of 5,5′-Dithiobis[succinimidyl(2-nitrobenzoate)]

[0178] 5,5′-dithiobis(2-nitrobenzoic acid) (50.0 mg, 0.126 mmol, AldrichChemical Company) and N-hyroxysuccinimide (29.0 mg, 0.252 mmol, AldrichChemical Company) were taken up in 1.0 mL dichloromethane.Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was added and the reactionmixture was stirred overnight at room temperature. After 16 hr, thereaction mixture was partitioned in EtOAc/H₂O. The organic layer waswashed 2×H₂O, 1×brine, dried (MgSO₄) and concentrated under reducedpressure. The residue was taken up in CH₂Cl₂, filtered, and purified byflash column chromatography on silica gel (130×30 mm, EtOAc:CH₂Cl₂ 1:9eluent) to afford 42 mg (56%)5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] as a white solid. H¹NMR(DMSO) ∂ 7.81-7.77 (d, 2H), 7.57-7.26 (m, 4H), 3.69 (s, 8 H).

Example 29 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-1,4-Bis(3-aminopropyl)piperazine Copolymer

[0179] 1,4-Bis(3-aminopropyl)piperazine (10 μL, 0.050 mmol, AldrichChemical Company) was taken up in 1.0 mL methanol and HCl (2 mL, 1 M inEt₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (30 mg, 0.050mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (35 μL, 0.20 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 23 mg (82%) of5,5′-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)piperazinecopolymer.

Example 30 Particle Sizing of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes

[0180] To 50 μg pDNA in 3 mL Ringers (0.85% sodium chloride, 0.03%potassium chloride, 0.03% calcium chloride) was added 170 μg5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazineCopolymer. Particle sizing (Brookhaven Instruments Coporation, ZetaPlusParticle Sizer, 190, 532 nm) indicated an effective diameter of 92 nmfor the complex. A 50 μg pDNA in 3 mL Ringers sample indicated noparticle formation.

[0181] 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-arninopropyl)piperazine Copolymer condenses pDNA,forming small particles.

Example 31 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicAcid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes

[0182] Four complexes were prepared as follows:

[0183] Complex I: pDNA (pCI Luc, 200 μg) in 1 mL H₂O and diluted with 9mL Ringers prior to injection.

[0184] Complex II: pDNA (pCI Luc, 200 μg) was mixed with poly-L-lysine(378 μg, MW 3400, Sigma Chemical Company) in 1 mL H₂O and diluted with 9mL Ringers prior to injection.

[0185] Complex III: pDNA (pCI Luc, 200 μg) was mixed with5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazineCopolymer (400 μg) in 1 mL H₂O and diluted with 9 mL Ringers prior toinjection.

[0186] Complex IV: pDNA (pCI Luc, 200 μg) was mixed with Histone H1 (1.2mg, Sigma Chemical Company) in 1 mL H₂O and diluted with 9 mL Ringersprior to injection.

[0187] 2.5 mL and 250 μL tail vein injections of the complex wereperformed (Zhang, G., Budker, V., Wolff, J, High Levels of Foreign GeneExpression in Hepatocytes from Tail Vein Injections of Naked PlasmidDNA. Human Gene Therapy, July, 1999, incorporated by reference). Resultsreported are for liver expression. Luciferase expression was determinedas previously reported (Wolff, J. A., Malone, R. W., Williams, P.,Chong, W., Acsadi, G., Jani, A. and Felgner, P.L. Direct gene transferinto mouse muscle in vivo. Science, 1465-1468,1990.). A Lumat LB 9507(EG&G Berthold, Bad-Wildbad, Germany) luminometer was used.

[0188] Results from 2.5 mL Injections

[0189] Complex I: 1,976,000

[0190] Complex II: 128,000

[0191] Complex III: 5,025,000

[0192] Complex IV: 1,960

[0193] Results from 250 μL Injections

[0194] Complex I: 985

[0195] Complex III: 1,140

[0196] Results indicate an increased level of luciferase expression inpDNA/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes over pCI LucDNA itself, pCI Luc DNA/poly-L-lysine complexes, and pCI Luc DNA/HistoneH1 complexes. These results also indicate that the pDNA is beingreleased from the pDNA/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes, and isaccessible for transcription.

[0197] 250 μL injection results were similar for bothpDNA/5,5′-Dithiobis(2-nitrobenzoic acid)1,4-Bis(3-aminopropyl)piperazine Copolymer complexes and pCI Luc DNA.

Example 32 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine-Tris(2-aminoethyl)amine Copolymer

[0198] 1,4-Bis(3-aminopropyl)piperazine (2.4 μL, 0.012 mmol, AldrichChemical Company) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol,Aldrich Chemical Company) were taken up in 0.5 mL methanol and HCl (1mL, 1 M in Et₂O, Aldrich Chemical Company) was added Et₂O was added andthe resulting HCl salt was collected by filtration.5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.016 mmol) wasadded and the mixture was taken up in 0.4 mL DMSO and 0.4 mL THF . Theresulting solution was stirred at room temperature anddiisopropylethylamine (5.9 μL, 0.042 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was diluted with 3 mL H₂O, anddialyzed in 12,000-14,000 MW cutoff tubing against water (2×2 L) for 48h. The solution was then removed from dialysis tubing and dried bylyophilization to yield 2.7 mg (30%) of 5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine-tris(2-aminoethyl)aminecopolymer.

Example 33 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine Copolymer

[0199] Tetraethylenepentamine (3.2 μL, 0.017 mmol, Aldrich ChemicalCompany) was taken up in 1.0 mL dichloromethane and HCl (1 mL, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg,0.017 mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.8 mg (62%) of5,5′-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine copolymer.

Example 34 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic Acid)-TetraethylenepentamineCopolymer Complexes

[0200] Complexes were prepared as follows:

[0201] Complex I: pDNA (pCI Luc, 200 μg) was added to 300μL DMSO then2.5 mL Ringers was added.

[0202] Complex II: pDNA (pCI Luc, 200 μg) was added to 300μL DMSO then5,5′-Dithiobis(2-nitrobenzoic acid)-Tetraethylenepentamine Copolymer(336 μg) was added followed by 2.5 mL Ringers.

[0203] 2.5 mL tail vain injections of the complex were performed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer intomouse muscle in vivo. Science, 1465-1468, 1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used.

[0204] 250 μL Injections

[0205] Complex I: 25,200,000

[0206] Complex II: 21,000,000

[0207] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic acid)-tetraethylenepentaminecopolymer complexes are nearly equivalent to pCI Luc DNA itself in 2.5mL injections. This indicates that the pDNA is being released from thecomplex and is accessible for transcription.

Example 35 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer

[0208] Tetraethylenepentamine (2.3 μL, 0.012 mmol, Aldrich ChemicalCompany) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, AldrichChemical Company) were taken up in 0.5 mL methanol and HCl (1 mL, 1 M inEt₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg,0.017 mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.9 mg (77%) of5,5′-dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine-tris(2-aminoethyl)amine copolymer.

Example 36 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicAcid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer Complexes

[0209] Complexes were prepared as follows:

[0210] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 mL Ringers was added.

[0211] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer (324 μg)was added followed by 2.5 mL Ringers.

[0212] 2.5 mL tail vain injections of the complex were preformed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer intomouse muscle in vivo. Science, 1465-1468,1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used.

[0213] 250 μL Injections

[0214] Complex 1: 25,200,000

[0215] Complex II: 37,200,000

[0216] pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer Complexesare more effective than pCI Luc DNA in 2.5 mL injections. Indicatingthat the pDNA is released from the complex and is accessible fortranscription.

Example 37 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer

[0217] N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.8 μL, 0.017 mmol,Aldrich Chemical Company) was taken up in 1.0 mL dichloromethane and HCl(1 mL, 1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was addedand the resulting HCl salt was collected by filtration. The salt wastaken up in 1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)](10 mg, 0.017 mmol) was added. The resulting solution was heated to 80°C. and diisopropylethylamine (12 μL, 0.068 mmol, Aldrich ChemicalCompany) was added by drops. After 16 hr, the solution was cooled,diluted with 3 mL H₂O, and dialyzed in 12,000-14,000 MW cutoff tubingagainst water (2×2 L) for 24 hr. The solution was then removed fromdialysis tubing and dried by lyophilization to yield 5.9 mg (66%) of5,5′-dithiobis(2-nitrobenzoicacid)-N,N′-bis(2-aminoethyl)-1,3-propanediamine Copolymer.

Example 38 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicAcid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer Complexes

[0218] Complexes were prepared as follows:

[0219] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 mL Ringers was added.

[0220] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (474 μg) wasadded followed by 2.5 mL Ringers.

[0221] Tail vain injections of 2.5 mL of the complex were preformed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported.

[0222] Results: 2.5 mL Injections

[0223] Complex 1: 25,200,000

[0224] Complex II: 341,000

[0225] pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine Copolymer Complexes provides luciferaseexpression indicating that the pDNA is being released from the complexand is accessible for transcription.

Example 39 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer

[0226] N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.0 μL, 0.012 mmol,Aldrich Chemical Company) and tris(2-aminoethyl)amine (0.51 μμL, 0.0034mmol, Aldrich Chemical Company) were taken up in 0.5 mL methanol and HCl(1 mL, 1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was addedand the resulting HCl salt was collected by filtration. The salt wastaken up in 1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)](10 mg, 0.017 mmol) was added. The resulting solution was heated to 80°C. and diisopropylethylamine (12 μL, 0.068 mmol, Aldrich ChemicalCompany) was added by drops. After 16 hr, the solution was cooled,diluted with 3 mL H₂O, and dialyzed in 12,000-14,000 MW cutoff tubingagainst water (2×2 L) for 24 hr. The solution was then removed fromdialysis tubing and dried by lyophilization to yield 6.0 mg (70%) of5,5′-dithiobis(2-nitrobenzoicacid)-N,N′-bis(2-aminoethyl)-1,3-propanediamine-tris(2-aminoethyl)aminecopolymer.

Example 40 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2- aminoethyl)amineCopolymer Complexes

[0227] Complexes were prepared as follows:

[0228] Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then2.5 mL Ringers was added.

[0229] Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer (474 μg) was added followed by 2.5 mL Ringers.

[0230] Tail vain injections of 2.5 mL of the complex were preformed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported.

[0231] Results: 2.5 mL Injections

[0232] Complex I: 25,200,000

[0233] Complex II: 1,440,000

[0234] Results indicate that pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer Complexes are less effective than pCI Luc DNA in 2.5 mLinjections. Although the complex was less effective, the luciferaseexpression indicates that the pDNA is being released from the complexand is accessible for transcription.

Example 41 Intramuscular Injections of Complexes from pDNA (pCILuc)/Physiologically Labile Disulfide Bond Containing Polymers on Mouse.

[0235] Seven complexes were prepared as follows:

[0236] Complex I: pDNA (pCI Luc, 40 μg) was added to 586 μL glucose (290mM)-HEPES (5 mM, pH 8).

[0237] Complex II: pDNA (pCI Luc, 40 μg) was added to 577 μL glucose(290 mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazineCopolymer (9 μL, 200 μg).

[0238] Complex III: pDNA (pCI Luc, 40 μg) was added to 573 μL glucose(290 mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazineCopolymer (13 μL, 200 μg).

[0239] Complex IV: pDNA (pCI Luc, 40 μg) was added to 574 pL glucose(290 mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoic acid)-Tetraethylenepentamine Copolymer (12μL, 70 μg).

[0240] Complex V: pDNA (pCI Luc, 40 μg) was added to 576 μL glucose (290mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer (10 μL,65 μg).

[0241] Complex VI: pDNA (pCI Luc, 40 μg) was added to 581 μL glucose(290 mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (5 μL, 94 μg).

[0242] Complex VII: pDNA (pCI Luc, 40 μg) was added to 570 μL glucose(290 mM)-HEPES (5 mM, pH 8). To this solution was added5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer (16 μL, 94 μg).

[0243] Direct muscle injections of 150 μL of the complex were preformedas previously described (See Budker, V., Zhang, G., Danko, I., Williams,P., and Wolff, J., “The Efficient Expression Of IntravascularlyDelivered DNA In Rat Muscle,” Gene Therapy 5, 272-6(1998); Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465-1468, 1990. which are incorporated herein by reference.). Sevendays post injection, the animals were sacrificed, and the muscleharvested. Samples were homogenized in lux buffer (I mL), andcentrifuged for 15 minutes at 4000 RPM. Luciferase expression wasdetermined as previously reported. Results reported for left quadracep:right quadracep (Complex IV-only injected into left quadracep).

[0244] Results:

[0245] Complex I: RLU 1,900: 4,316

[0246] Complex II: RLU 13,433: 20,640

[0247] Complex III: RLU 10,156 : 39,491

[0248] Complex IV: RLU 9,888:

[0249] Complex V: RLU=19,565: 5,806

[0250] Complex VI: RLU =270: 427

[0251] Complex VII: RLU =973: 6,000

[0252] The complexes prepared from pCI Luc DNA/Physiologically LabileDisulfide Bond Containing Polymers are effective in direct muscleinjections. The luciferase expression indicates that the pDNA is beingreleased from the complex and is accessible for transcription. Complexesprepared with 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer were the mosteffective, giving luciferase expression levels 2 to 10 times as high aspDNA.

Example 42 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-Pentaethylenehexamine Copolymer

[0253] Pentaethylenehexamine (4.2 μL, 0.017 mmol, Aldrich ChemicalCompany) was taken up in 1.0 mL dichloromethane and HCl (1 mL, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.9 mg (58%) of5,5′-dithiobis(2-nitrobenzoic acid)-pentaethylenehexamine Copolymer.

Example 43 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-Pentaethylenehexamine-Tris(2-aminoethyl)amine Copolymer

[0254] Pentaethylenehexamine (2.9 μL, 0.012 mmol, Aldrich ChemicalCompany) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, AldrichChemical Company) were taken up in 0.5 mL methanol and HCl (1 mL, 1 M inEt₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg,0.017mmol) was added. The resulting solution was heated to 80° C. anddisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.0 mg (64%) of5,5′-dithiobis(2-nitrobenzoicacid)-pentaethylenehexamine-tris(2-aminoethyl)amine copolymer.

Example 44 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicAcid)-N-(3-Aminopropyl)-1,3-propanediamine Copolymer

[0255] 5,5′-Dithiobis[succinimidyl(2-nitrobenzoate)] (2.5 mg, 0.0042mmol) was taken up in 10 μL of DMF. N-(3-aminopropyl)-1,3-propanediamine(0.6 μL, 0.004 mmol, Aldrich Chemical Company) was added with 10 μLHEPES 250 mM, pH 7.5. After 1 hr the solution was concentrated underreduced pressure. The resulting residue was dissolved in 0.42 mL DMSO.Analysis of the solution on SDS-PAGE versus poly-L-lysisne hydrobromide(MW of 1000, 7500, 15000) indicated an approximate molecular weightrange of 3500-8000 for the polymer.

Example 45 Synthesis of 5,5′-dithiobis(2-nitrobenzoicAcid)-1,4-bis(3-aminopropyl)piperazine-Folate Copolymer

[0256] Folate-PEG(3400 MW)-NH2 was prepared according to the knownprocedure (Lee, R. J., Low, P. S. Biochimica et Biophysica Acta 1233,1995, 134-144). Folate-PEG-NH2 was acylated with succinylatedN-(3-(BOC)aminopropyl)-1,3-propaneamine(BOC)amine. Removal of the BOCprotecting groups afforded the Folate monomer.1,4-bis(3-aminopropyl)piperazine (5.0 μL, 0.023 mmol, Aldrich ChemicalCompany) and folate monomer (5.0 mg, 0.0012 mmol) were taken up in 0.4mL methanol and HCl (1 mL, 1 M in Et₂O, Aldrich Chemical Company) wasadded. The resulting suspension was concentrated under reduced pressureto afford a white solid. The salt was taken up in 0.5 mL DMF and5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (14 mg, 0.025 mmol) wasadded. The resulting solution was heated to 80° C. anddiisopropylethylamine (18 μL, 0.10 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000-14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 13 mg (68%) of5,5′-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)piperazine-folate copolymer.

Example 46 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer

[0257] H₂N-EEEEEEEE-NHCH₂CH₂NH₂ (5.0 mg, 0.0052 mmol, Genosis) was takenup in 0.1 mL HEPES (250 mM, pH 7.5).5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (3.1 mg, 0.0052) was addedwith 0.2 mL DMSO and the mixture was stirred overnight at roomtemperature. After 16 hr the solution was heated to 70° C. for 10 min,cooled to room temperature and diluted to 1.10 mL with DMSO.

Example 47 Complex Formation with 5,5′-Dithiobis(2-nitrobenzoicacid)-PolyGlutamicacid (8mer) Copolymer

[0258] Fluorescein labeled DNA was used for the determination of DNAcondensation in complexes with 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer. pDNA was modified to a levelof 1 fluorescein per 20 bases using Mirus' LabellT™ Fluorescein kit. Thefluorescence was determined using a fluorescence spectrophotometer(Shimadzo RF-1501 Fluorescence Spectrophotometer), at an excitationwavelength of 497 nm, and an emission wavelength of 520 nm.

[0259] To fluorescein labeled DNA (10 μg) in 1 mL HEPES (25 mM, pH 7.5)was added polyornithine (18 μg, Sigma Chemical Company). The mixtureswere held at room temperature for 5 minutes and the fluorescence wasdetermined. (see: Trubetskoy, V. S., Slattum, P. M., Hagstrom, J. E.,Wolff, J. A., Budker, V. G., “Quantitative Assessment of DNACondensation,” Anal. Biochem (1999) incorporated by reference) Sincefluorescence intensity is decreased by DNA condensation, resultsindicate that polyornithine compacts DNA. To the resulting complex wasadded 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)Copolymer (60 μg), and the fluorescence was again determined. Thefluorescence of the sample decreased further.

[0260] Upon the addition of 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer to the sample, the fluorescencedecreased, indicating the formation a triple complex. No competition ofthe 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)Copolymer for the polyornithine was observed (increase in fluorescence).

Example 48 Transfection of 3T3 Cells with 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer

[0261] Three complexes were formed:

[0262] Complex I) To 300 μL Opti-MEM was added LT-1TM (12 μg, MirusCorporation) followed by pDNA (pCI Luc, 4 μg).

[0263] Complex II) To 300 μL Opti-MEM was added LT-1™ (12 μg, MirusCorporation) followed by pDNA (pCI Luc, 4 μg), and5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer) Copolymer(4 μg).

[0264] Complex III) To 300 μL Opti-MEM was added LT-1™ (12 μg, MirusCorporation) followed by pDNA (pCI Luc, 4 μg), and Poly-Glutamicacid (4μg, MW 49000, Sigma Chemical Company).

[0265] Transfections were carried out in 35 mm wells. At the time oftransfection, 3T3 cells, at approximately 50% confluency, were washedonce with PBS (phosphate buffered saline), and subsequently stored inserum-free media (2.0 mL, Opti-MEM, Gibco BRL). 150 μL of complex wasadded to each well. After a 3.25 h incubation period at 37° C., themedia containing the complexes was aspirated from the cells, andreplaced with complete growth media, DMEM with 10% fetal bovine serum(Sigma). After an additional incubation of 48 hours, the cells wereharvested and the lysate was assayed for luciferase expression aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer intomouse muscle in vivo. Science, 1465-1468,1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used.

[0266] Results:

[0267] Complex I: RLU =17,000,000

[0268] Complex II: RLU =14,000,000

[0269] Complex III: RLU =26,000,000

[0270] The addition of Poly-Glutamicacid (4 μg, MW 49000, Sigma ChemicalCompany) in the transfection experiment improved the pDNA expression.The addition of 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid(8mer) Copolymer (4 μg) while not improving the pDNA expression was notdetrimental to the expression.

Example 49 Demonstration of Reduction of by5,5′-dithiobis(2-nitrobenzoic acid)-Containing Copolymers by Glutathione

[0271] To a solution of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (100 μg) in 0.5 mLHEPES (25 mM, pH 8) was added glutathione (final concentration of 2 mM).The absorbance of the sample was measured at λ 412 (The cleaveddisulfide has an absorbance maximum at λ 412. See Hermanson, G. T.Bioconjugate Techniques, Academic Press, New York, New York, 1996, pp88) versus time (Beckman DU-7 Spectrophotometer).

[0272] To a solution of 5,5′-dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine copolymer (50 μg) in 0.5 mL HEPES (25 mM,pH 8) was added glutathione (final concentration of 2 mM). Theabsorbance of the sample was measured at λ 412 (The cleaved disulfidehas an absorbance maximum at λ 412. See Hermanson, G. T. BioconjugateTechniques, Academic Press, New York, New York, 1996, pp 88) versus time(Beckman DU-7 Spectrophotometer).

[0273] To a solution of 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer (50 μg) in 0.5 mL HEPES (25 mM,pH 8) was added glutathione (final concentration of 2 mM). Theabsorbance of the sample was measured at λ 412 (The cleaved disulfidehas an absorbance maximum at λ 412. See Hermanson, G. T. BioconjugateTechniques, Academic Press, New York, New York, 1996, pp 88) versus time(Beckman DU-7 Spectrophotometer).

[0274] Each sample showed a rapid increase in the absorbance at λ 412upon the addition of glutathione, indicating cleavage of the disulfidebond. Half life values were estimated as:

[0275] 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer t_(½)=42 sec.

[0276] 5,5′-dithiobis(2-nitrobenzoic acid)-tetraethylenepentaminecopolymer t_(½)=75 sec.

[0277] 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)Copolymer t_(½)=24 sec.

[0278] The experiment demonstrates rapid cleavage of the disulfide bondof 5,5′-dithiobis(2-nitrobenzoic acid)-containing copolymers with thephysiological reducing agent glutathione.

Example 50 Analysis of Delivery to Cells by VP22 Peptide:

[0279] Grow HeLa cells on glass coverslips by incubating at 4° C. inDelbecco's Modified Eagle's Media (DMEM) supplemented with 50 μg VP22peptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external VP22-fluorophore.Remove the media and then either process cells for fluorescencemicroscopy or incubate three more hours at 4° C. with DMEM with mediachanges every hour (chase). The cells that are chased are then processedfor fluorescence microscopy. Cells processed for fluorescence microscopyare washed 3× in phosphate-buffered saline (PBS), fixed in PBS+4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides. The presence of fluorophore is detected by confocal microscopy(Zeiss LSM 510).

Example 51 Analysis of Delivery to Cells by ANTP Peptide:

[0280] Grow HeLa cells on glass coverslips by incubating at 4° C. inDelbecco's Modified Eagle's Media (DMEM) supplemented with 50 μg ANTPpeptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external ANTP-fluorophore.Remove the media and then either process cells for fluorescencemicroscopy or incubate three more hours at 4° C. with DMEM with mediachanges every hour (chase). The cells that are chased are then processedfor fluorescence microscopy. Cells processed for fluorescence microscopyare washed 3× in phosphate-buffered saline (PBS), fixed in PBS+4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides. The presence of fluorophore is detected by confocal microscopy(Zeiss LSM 510).

[0281] The foregoing is considered as illustrative only of theprinciples of the invention. Furthermore, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described. Therefore, all suitable modifications andequivalents fall within the scope of the invention.

We claim:
 1. A compound for inserting into an organism, comprising: thecompound having a disulfide bond that is labile under physiologicconditions selected from the group consisting of (a) a disulfide bondthat is cleaved more rapidly than oxidized glutathione and (b) adisulfide bond constructed from thiols in which one of the constituentthiols has a lower pKa than glutathione and (c) a disulfide bond that isactivated by intramolecular attack from a free thiol wherein thecompound contains a transduction signal.
 2. The compound of claim 1wherein the transduction signal consists of Tat.
 3. The compound ofclaim 1 wherein the transduction signal consists of VP22.
 4. Thecompound of claim 1 wherein the transduction signal consists of ANTP. 5.The compound of claim 1 wherein the transduction signal consists of apolymer containing a cationic charge.
 6. The compound of claim 5 claim 1wherein the transduction signal consists of a peptide containingcationic residues.
 7. A process for delivering a compound having alabile disulfide bond into a mammal, comprising: a) forming the compoundhaving a disulfide bond selected from the group consisting of (i) adisulfide bond that is cleaved more rapidly than oxidized glutathione,and (ii) a disulfide bond constructed from thiols in which one of theconstituent thiols has a lower pKa than glutathione, and (iii) adisulfide bond that is activated by intramolecular attack from a freethiol; b) attaching a transduction signal to the compound; c) insertingthe compound into the mammal; and, d) releasing the bond between thesulfur atoms in the disulfide.
 8. The process of claim 7 wherein thetransduction signal consists of Tat.
 9. The process of claim 7 whereinthe transduction signal consists of VP22.
 10. The process of claim 7wherein the transduction signal consists of ANTP.
 11. The process ofclaim 7 wherein the transduction signal consists of a peptide containinga cationic charge.
 12. The process of claim 11 wherein the transductionsignal consists of a peptide containing cationic residues.
 13. Thecompound of claim 1 wherein the compound consists of nucleic acids.